In 1990, Wright et al. elucidated that a 53 kDa glycoprotein named CD14 expressed on the membrane of a monocyte is a receptor for LPS, which is an endotoxin (Science 249: p.1431, 1990). The CD14 protein includes a soluble CD14 protein (hereinafter, also referred to as sCD14 protein or soluble CD14 protein) in addition to a membrane bound type CD14 protein (hereinafter, also referred to as mCD14 protein). It has been reported that in blood there are a plurality of soluble CD14 proteins differing in molecular weight (Labeta MO, Eur. J. Immunol., 23, 2144, 1993). The soluble CD14 proteins were considered to be CD14 proteins on the membrane that were released due to the activation of the cell. On the contrary, it has recently been reported that mCD14 protein is expressed on primary human liver cells and liver cell lines (Su GL, J. Hematology, 31, 435, 1999). In a transgenic mouse in which an 80 kb genomic human CD14 protein containing the 5′ and 3′ flanking regions of the gene of CD14 protein were introduced, mCD14 proteins were expressed not only in monocytes/macrophages but also in liver cells (Hetherlington C, et al., J. Immunol., 162, 503, 1999). Also, it has hitherto been considered that sCD14 proteins trap LPS in the blood and transport it to HDL so that clearance of LPS is made in the liver (J. Exp. Med., 181, 1743, 1995).
Thus, as its physiological function, while the soluble CD14 protein directly or indirectly clears LPS, a complex of LPS and sCD14 protein bound thereto (sCD14 protein/LPS) considerably activates macrophages through mCD14 proteins on the macrophages to induce inflammatory cytokines (Hailmann, E. et al., J. Immunol., 156, 4384, 1996). Further, the sCD14 protein/LPS induces cellular death of vascular endothelial cells or vascular smooth muscle cells and production of inflammatory cytokines (Loppnow, H. et al., Infection & Immunity, 63, 1020, 1995). The vascular endothelial cells and vascular smooth muscle cells were confirmed to have no mCD14 protein, so that existence of a receptor that recognizes sCD14 protein/LPS was suggested (Tobias et al., J. Exp. Med., 178, 2193, 1993). Also, there is a report that the endothelial cells of a patient with hemoglobin nocturnal enuresis (PNH) who is suffering from GPI biosynthesis failure has an LPS response with a sCD14 protein (J. Lab. Clin. Med., 125, 662, 1995).
Recently, it has been found that CD14 proteins recognize phosphatidylserine and contributes to the removal of cells that caused apoptosis. Thus a function of CD14 protein other than that as LPS receptor has been elucidated (Devitt A, Nature, 392, 505, 1998). Moreover M. Labeta et al. reported that sCD14 proteins have an activity of suppressing T cell activator (M. Labeta, et al., Eur. J. Immunol. 29, 265, 1999).
Reportedly, soluble CD14 proteins increase in sera or urines of patients with sepsis, external injuries, burn injuries, or rheumatism and are considered to participate in the exclusion of LPS or in LPS signal transfer. Landmann et al. conducted Western analysis of soluble CD14 proteins of the serum from a patient with sepsis and reported that a high molecular weight soluble CD14 protein is at high levels in death cases from sepsis or patients with paroxysmal nocturnal hemoglobinuria (PNH) and that in normal sera, there was detected no high molecular weight soluble CD14 protein that was detected in death cases from sepsis (The Journal of Infectious Disease, Vol. 171, p. 639, 1995). Existence of the subtypes differing in molecular weight is attributable to a difference in glyco chain and after the removal of N and 0 bound type glyco chain, there are still two kinds of soluble CD14 proteins differing in molecular weight as reported by Stelter et al. (Eur. J. Biochem., Vol. 236, p.457, 1996). Buffler et al. performed C-terminus analysis of a soluble CD14 protein and reported that a GPI group binds to the serine residue at the 327th position of the soluble CD14 protein and that a soluble CD14 protein having a molecular weight of about 56 kDa is a molecule species that is not GPI anchored (Eur. J. Immunol., Vol. 25, p.604, 1995).
As described above, given that there is such a report that a subtype of a high molecular weight soluble CD14 protein in blood is at high levels in sera of patients suffering from sepsis in severe cases, it is suggested that measurement of soluble CD14 proteins will be clinically important. However, these analyses have the problem that they are by a Western method, which requires complicated operational process and is low in sensitivity, thus they are impractical. That is, since there has been no specific measurement method that is highly sensitive but simple and easy, clinical usefulness of measurement of soluble CD14 proteins could not be proved. Therefore, to prove the clinical usefulness of high molecular weight soluble CD14 proteins, development of a method for measuring them with high sensitivity, simply and easily, and specifically has been required.
As antibodies to soluble CD14 proteins, there have been prepared many anti-CD14 protein antibodies in addition to MEM-18 prepared by Bazil et al. (Eur. J. Imunol., Vol. 16, p.1583, 1986), RoMo-1 prepared by Shutt et al. (Allerg. Immunol. (Leipz), Vol. 34, No. 1, p.17, 1988), 3C10 prepared by Steinman et al. (J. Exp. Med., Vol. 158, No. 1, p.126, 1983). Published Translation of Japanese Patent Application No. Hei 8-510909 discloses 28C5, 23G4 and the like.
It has been elucidated that 3C10, MEM-18, 28C5 or 23G4 has an activity of inhibiting the binding of LPS to a CD14 protein. Particularly, 3C10 and MEM-18 have their respective binding regions on the side of amino termini, that is, in the amino acids 7-14, (J. Biol. Chem., Vol. 270, No. 29, p. 17237, 1995) and amino acids 57-64 (J. Biol. Chem., Vol. 270, No. 10, p. 5219, 1995) described in SEQ ID No. 1, which is the binding region for LPS. Also, a system for measuring soluble CD14 proteins using the antibodies (J. Immunol. Methods, Vol. 155, p. 225, 1992) has been prepared. Since the measurement systems using the antibodies use antibodies that recognize amino termini, the soluble CD14 proteins in the blood to be measured are all the soluble CD14 proteins that exist in blood (hereinafter, the soluble CD14 proteins measured with an antibody having specificity to the N-terminus are described as total amount). In other words, the antibodies to soluble CD14 proteins thus far reported recognize all the soluble CD14 proteins that are present in blood but do not specifically recognize high molecular weight soluble CD14 proteins, so that it is impossible to perform specific measurement of the high molecular weight soluble CD14 proteins using the antibodies.
Further, the present inventors have reported in Japanese Patent Application No. 2000-099617 that soluble CD14 protein (1-285) and low molecular weight soluble CD14 36 kDa protein have an activity of inhibiting the induction of inflammatory cytokines. It is considered that while the low molecular weight soluble CD14 36 kDa protein in blood directly or indirectly clears LPS as its physiological function, it also inhibits a complex of LPS and sCD14 protein bound thereto (sCD14 protein/LPS) from considerably activating macrophages through mCD14 proteins on the macrophages and inducing inflammatory cytokines. Further, since the sCD14 protein/LPS induces cellular death of vascular endothelial cells or vascular smooth muscle cells and production of inflammatory cytokines, the low molecular weight soluble CD14 36 kDa protein is considered to similarly inhibit the injuries of vascular endothelial cells and vascular smooth muscle cells. From the above, it is presumed that in patients suffering from sepsis, a mechanism exists, in which LPS stimulation enhances production of mCD14 protein on macrophages and thereafter the mCD14 protein is severed with protease to produce a low molecular weight soluble CD14 protein and the low molecular weight soluble CD14 protein is expected to work as a defense factor of an organism for inhibiting injuries of cells. Therefore, it is considered that an increase in the low molecular weight soluble CD14 protein (36 kDa) in patients suffering sepsis is a defense mechanism in an organism that is initiated by LPS stimulation, collapse of which mechanism aggravates the state of patients suffering from sepsis.
In other words, it is suggested that, like the measurement of the high molecular weight soluble CD14 proteins, measurement of the low molecular weight soluble CD14 proteins is also clinically important. However, the antibodies to soluble CD14 proteins thus far reported fail to specifically recognize the low molecular weight soluble CD14 proteins either. Moreover, although details will be described in DISCLOSURE OF THE INVENTION below, it is even more difficult to prepare antibodies that specifically recognize the low molecular weight soluble CD14 proteins than antibodies that specifically recognize the high molecular weight soluble CD14 proteins. For this reason, development of a method for measuring a low molecular weight soluble CD14 protein with high sensitivity, simply and easily and specifically has been demanded.